Catalysis of the Photo-Fries Reaction: Antibody-Mediated Stabilization of High Energy States

Article abstract of DOI:10.1021/ja038016n

A conformationally constrained hapten is presented that is capable of catalyzing the first antibody-mediated photo-Fries rearrangement. In this reaction, absorption of light energy by a diphenyl ether substrate results in homolytic C-O bond cleavage followed by recombination to yield biphenyl-derived products. The most proficient antibody studied converts 4-phenoxyaniline 15 into 2-hydroxy-5-aminobiphenyl 16 under high-intensity irradiation at a rate of 8.6 μM/min. These results support a recent hypothesis stating that immunization with conformationally constrained haptens provides higher titers for the acquisition of simple binding antibodies; however, in this case, conformational constraint does not ensure the development of more efficient catalysts. Using the obtained antibodies, the presence of products resulting from escape of free radicals from the solvent cage can be suppressed, altering the excited state energy surface such that free radicals are funneled into the formation of the desired biphenyl product. However, studies also show the inactivation of the antibodies as a result of photodecay of the biphenyl product. Using an isocyanate scavenging resin, the photodecay product could be removed and the inactivation of the antibody drastically reduced. Furthermore, despite the observed photodecay, turnover of the antibody was present; this represents the first case in which true turnover of a photochemical reaction using a catalytic antibody could be observed.

Full text of DOI:10.1021/ja038016n

Published on Web 11/20/2003
Catalysis of the Photo-Fries Reaction: Antibody-Mediated
Stabilization of High Energy States
Tobin J. Dickerson, Martin R. Tremblay, Timothy Z. Hoffman, Diana I. Ruiz, and
Kim D. Janda*
Contribution from the Department of Chemistry and The Skaggs Institute for Chemical Biology,
The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037
Received August 19, 2003; E-mail: kdjanda@scripps.edu
Abstract: A conformationally constrained hapten is presented that is capable of catalyzing the first antibody-
mediated photo-Fries rearrangement. In this reaction, absorption of light energy by a diphenyl ether substrate
results in homolytic C-O bond cleavage followed by recombination to yield biphenyl-derived products.
The most proficient antibody studied converts 4-phenoxyaniline 15 into 2-hydroxy-5-aminobiphenyl 16 under
high-intensity irradiation at a rate of 8.6 µM/min. These results support a recent hypothesis stating that
immunization with conformationally constrained haptens provides higher titers for the acquisition of simple
binding antibodies; however, in this case, conformational constraint does not ensure the development of
more efficient catalysts. Using the obtained antibodies, the presence of products resulting from escape of
free radicals from the solvent cage can be suppressed, altering the excited state energy surface such that
free radicals are funneled into the formation of the desired biphenyl product. However, studies also show
the inactivation of the antibodies as a result of photodecay of the biphenyl product. Using an isocyanate
scavenging resin, the photodecay product could be removed and the inactivation of the antibody drastically
reduced. Furthermore, despite the observed photodecay, turnover of the antibody was present; this
represents the first case in which true turnover of a photochemical reaction using a catalytic antibody could
be observed.
Introduction
It is widely accepted that the photo-Fries reaction proceeds
through radical pairs; however little physical evidence has been
A number of rearrangements of aromatic compounds are
known including the Claisen rearrangement,1 the Fries rear-
rangement,2 and the benzidine rearrangement.3 Each of these
reactions can proceed via two distinct and readily different-
iated mechanisms, photochemical and acid-catalyzed rear-
rangement. Although diphenyl ethers such as 1 have been
shown to be stable to acids and high temperatures, upon UV
irradiation these compounds can be converted into products 2-5,
albeit in low quantum yield (Scheme 1). As a result of this low
reactivity coupled with limited synthetic utility, this reaction
has only been sparingly studied in the greater than fifty years
since its discovery.4 Formally, this reaction is a photo-Claisen
rearrangement, albeit distinct from the [3,3] photo-Claisen
rearrangement.
reported. A report by Haga studied this process in detail, and a
number of interesting features were uncovered.5 Mechanistically,
it has been suggested that the reaction occurs by excitation of
1 from the ground state to a singlet state, followed by homolytic
cleavage of the C-O bond to give a singlet radical pair.
Recombination and aromatization of these radical pairs leads
to some of the observed products, while others result from
dissociation of the radical pairs to free radicals and hydrogen
abstraction from the solvent. Asymmetric ethers having their
para position occupied by a substituent were also studied, and
it was found that the product distribution was heavily influenced
by the electronic character of this substitutent (Scheme 2).
Specifically, electron-donating substituents predominantly gave
products resulting from path a, while electron-withdrawing
substituents gave products exclusively from path b. Amazingly,
despite the radical nature of this reaction, the preference of the
mechanism for aryloxy-phenyl cleavage with electron-donating
substitutent and aryl-phenoxy cleavage with electron-with-
drawing substituents correlated with the Hammett σ values.
(1) de Mayo, P. D. Molecular Rearrangements; John Wiley & Sons: New
York, 1963; Vol. 1, pp 660-684.
(2) (a) Blatt, A. H. Organic Reactions; John Wiley & Sons: New York, 1942;
Vol. 8, pp 342-384. (b) Bellus, D. AdVances in Photochemistry; John Wiley
& Sons: New York, 1971; Vol. 1, pp 109-159.
(3) Shine, H. J. Aromatic Rearrangements; Elsevier: Amsterdam, 1967; pp
124-179.
To effectively exploit the energy potential provided by light,
nature has evolved particularly efficient mechanisms to both
harness and direct light-based energy. Tantamount among these
(4) (a) Kelly, D. P.; Pinhey, J. T.; Rigby, R. D. G. Aust. J. Chem. 1969, 22,
977-979. (b) Hageman, H. J.; Louwerse, H. L.; Mijs, W. J. Tetrahedron
1970, 26, 2045-2052. (c) Ogata, Y.; Takagi, K.; Ishino, I. Tetrahedron
1970, 26, 2703-2709. (d) Jochek, H.-I.; Miller, S. I. J. Am. Chem. Soc.
1996, 88, 3269-3272. (e) Hageman, H. J.; Huysmans, W. G. B. Recl. TraV.
Chim. Pays.-Bas. 1972, 91, 528-532. (f) Elix, J. A.; Murphy, D. P. Aust.
J. Chem. 1975, 28, 1559-1582.
(5) Haga, N.; Takayanagi, H. J. Am. Chem. Soc. 1996, 61, 735-745.
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J. AM. CHEM. SOC. 2003, 125, 15395-15401
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A R T I C L E S
Dickerson et al.
Scheme 1. Photo-Fries Rearrangement of a Diphenyl Ether and Observed Reaction Products
Scheme 2. Photo-Fries Rearrangement of Asymmetric Diphenyl Ethersa
a When X ) EDG (e.g., OMe, OH, NH2, CH3), path a . path b. When X ) EWG (e.g., CO2Me, CN), path a , path b.
processes are the rhodopsin-activated enzymatic cascade in
vision,6 the electron-transfer events present in photosynthesis,7
and the fragmentation of thymine dimers in DNA repair.8 These
enzymes have evolved in such a way that high energy states
are processed correctly to produce only the desired chemical
outcome. Thus, a critical challenge in the production of
photochemical catalysts is not only the stabilization of a given
transition state, but also rigorous control over the high-energy
intermediates produced along the reaction coordinate.
including H2O2, H2O3, and ozone from singlet oxygen clearly
shows that the antibody structure is surprisingly resilient to a
variety of highly reactive chemical species.15 The vast majority
of reported antibody-catalyzed reactions are thermal in nature,
and in fact, there are no examples of photochemical reactions
demonstrating uninterrupted antibody turnover. However, ex-
amples do exist of antibody accelerated/energy altered photo-
chemical reactions including Norrish type II reactions,16 pyri-
midine dimer cleavage,17 and the production of an antibody-
stilbene “exciplex” which productively funnels energy into a
long-lived blue fluorescent species.18 Little is known regarding
the chemical structure of photochemical transition states and
corresponding stable analogues; thus, in all reported antibody
photochemical reactions, haptens have been designed which
directly resemble ground-state species.
Alternative protein-based entities that can provide mechanistic
insight into biochemical processes include catalytic antibodies.
The development of the first catalytic antibodies in 1986 helped
galvanize new research opportunities into the creation of de noVo
biocatalysts.9 Quickly following this seminal discovery, it was
realized that the much of the true power of catalytic antibodies
was in the catalysis of reactions which have no known natural
counterpart.10 In this context, numerous antibody catalysts have
since been identified which can catalyze reactions such as
Diels-Alder and [1.3]-dipolar cycladditions,11 anti-Baldwin ring
closure reactions,12 cationic cyclizations,13 and the Bergman
cyclization of enediynes.14 Furthermore, the recent demonstra-
tion that all antibodies can produce reactive oxygen species
In this study, we report the development of the first antibodies
that are capable of catalyzing the Fries photochemical rear-
rangement of diphenyl ethers. By not only affecting the energy
surface of the ground state but also altering the surface of the
excited state, these antibodies accelerate the formation of the
desired photochemical product while also drastically reducing
the proportion of byproducts produced as a result of escape of
radicals from the solvent cage. This study also demonstrates
that antibodies can turnover substrates for photochemical
reactions under standard conditions used for studying reaction
(6) Stryer, L. Cold Spring Harbor Symp. Quantum Biol. 1988, 53, 283-294
and references therein.
(7) Deisenhofer, J.; Michel, H.; Huber, R. Trends Biochem. Sci. 1985, 10, 243-
248 and references therein.
(8) Begley, T. P. Acc. Chem. Res. 1994, 27, 394-401 and references therein
(9) (a) Tramantano, A.; Janda, K. D.; Lerner, R. A. Science 1986, 234, 1566-
1570. (b) Pollack, S. J.; Jacobs, J. W.; Schultz, P. G. Science 1986, 234,
1570-1573. (c) Wentworth, P., Jr.; Janda, K. D. Curr. Opin. Chem. Biol.
1998, 2, 138-144.
(10) Wentworth, P., Jr.; Janda, K. D. Cell Biochem. Biophys. 2001, 35, 63-87.
(11) (a) Gouverneur, V. E.; Houk, K. N.; de Pascual-Teresa, B.; Beno, B.; Janda,
K. D.; Lerner, R. A. Science 1993, 262, 204-208. (b) Yli-Kauhaluoma, J.
T.; Ashley, J. A.; Lo, C.-H.; Tucker, L.; Wolfe, M. M.; Janda, K. D. J.
Am. Chem. Soc. 1995, 117, 7041-7047. (c) Toker, J. D.; Wentworth, P.,
Jr.; Hu, Y.; Houk, K. N.; Janda, K. D. J. Am. Chem. Soc. 2000, 122, 3244-
3245.
(12) (a) Janda, K. D.; Shevlin, C. G.; Lerner, R. A. Science 1993, 259, 490-
493. (b) Janda, K. D.; Shevlin, C. G.; Lerner, R. A. J. Am. Chem. Soc.
1995, 117, 2659-2660
(13) (a) Li, T.; Janda, K. D.; Ashley, J. A.; Lerner, R. A. Science 1994, 264,
1289-1293. (b) Li, T.; Hilton, S.; Janda, K. D. J. Am. Chem. Soc. 1995,
117, 3308-3309. (c) Li, T.; Janda, K. D.; Lerner, R. A. Nature 1996, 379,
326-327. (d) Li, T.; Lerner, R. A.; Janda, K. D. Acc. Chem. Res. 1997,
30, 115-121. (e) Hasserodt, J.; Janda, K. D.; Lerner, R. A. J. Am. Chem.
Soc. 1997, 119, 5993-5998. (f) Hasserodt, J.; Janda, K. D.; Lerner, R. A.
J. Am. Chem. Soc. 2000, 122, 40-45.
(14) Jones, L. H.; Harwig, C. W.; Wentworth, P., Jr.; Simeonov, A.; Wentworth,
A. D.; Py, S.; Ashley, J. A.; Lerner, R. A.; Janda, K. D. J. Am. Chem. Soc.
2001, 123, 3607-3608.
(15) (a) Wentworth, A. D.; Jones, L. H.; Wentworth, P., Jr.; Janda, K. D.; Lerner,
R. A. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 10930-10935. (b) Wentworth,
P., Jr.; Jones, L. H.; Wentworth, A. D.; Zhu, X.; Larsen, N. A.; Wilson, I.
A.; Xu, X.; Goddard, W. A., III; Janda, K. D.; Eschenmoser, A.; Lerner,
R. A. Science 2001, 293, 1806-1811. (c) Wentworth, P., Jr.; McDunn, J.
E.; Wentworth, A. D.; Takeuchi, C.; Nieva, J.; Jones, T.; Bautista, C.; Ruedi,
J. M.; Gutierrez, A.; Janda, K. D.; Babior, B. M.; Eschenmoser, A.; Lerner,
R. A. Science 2002, 298, 2195-2199.
(16) (a) Taylor, M. J.; Hoffman, T. Z.; Yli-Kauhaluoma, J. T.; Lerner, R. A.;
Janda, K. D. J. Am. Chem. Soc. 1998, 120, 12783-12790. (b) Saphier, S.;
Sinha, S. C.; Keinan, E. Angew. Chem., Int. Ed. 2003, 42, 1378-1381.
(17) (a) Cochran, A. G.; Sugasawara, R.; Schultz, P. G. J. Am. Chem. Soc. 1988,
110, 7888-7890. (b) Jacobsen, J. R.; Cochran, A. G.; Stephans, J. C.; King,
D. S.; Schultz, P. G. J. Am. Chem. Soc. 1995, 117, 5453-5461.
(18) Simeonov, A.; Matsushita, M.; Juban, E. A.; Thompson, E. H. Z.; Hoffman,
T. Z.; Beuscher, A. E., IV; Taylor, M. J.; Wirsching, P.; Rettig, W.;
McCusker, J. K.; Stevens, R. C.; Millar, D. P.; Schultz, P. G.; Lerner, R.
A.; Janda, K. D. Science 2000, 290, 307-313.
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Catalysis of Antibody-Mediated Photo-Fries Reaction
A R T I C L E S
Scheme 3. Synthesis of Haptens Used to Elicit Catalytic
hemocyanin (KLH) and bovine serum albumin (BSA) using
standard protocols.10 Two panels of 2 and 14 monoclonal
antibodies obtained from immunization with hapten 12 and 13,
respectively, were purified20 and screened under described
conditions (vide supra). The most congruent substrate to haptens
12 and 13 was deemed to be 14, wherein the linker has been
replaced with an acetamide functionality. High-intensity, broad
band UV irradiation of substrate 14 under the assay conditions
(50 mM phosphate-buffered saline, pH 7.4, 2% acetonitrile)
revealed that extended reaction times (>12 h) were necessary
in order to achieve significant production of product. These
extended reaction times are presumed to be due to the relatively
minor effect an acetamido functionality has on the electronic
character of the phenyl ring (σpara ) -0.01). In previous
experiments, both strongly donating and strongly withdrawing
substituents decreased the necessary irradiation times.5 Unfor-
tunately, although six-members of the antibody panel showed
increased rates of product formation over background, the
extended irradiation times necessary (>12 h) also caused
antibody precipitation and an inability to measure kinetics. We
envisioned that the obtained antibodies might also bind the close
homologue 4-phenoxyaniline 15, a compound which is expected
to react significantly faster in this reaction.5 Irradiation of 15
under the assay conditions yielded primarily biphenyl product
16 corresponding to homolytic cleavage C-O from path a
(Scheme 4), in agreement with previous studies. From the
obtained panels of antibodies, six of the mAbs raised against
hapten 13 (MT4-1G11, 3G2, 5A1, 6F11, 7E4, and 8D5) and
one mAb raised against hapten 12 (MT2-21C4) showed
enhanced formation of 16 relative to uncatalyzed rates. Of these,
two mAbs (MT4-3G2 and MT2-21C4) that showed the greatest
enhancement were chosen for further study. Interestingly, both
haptens led to products resulting from path a, implying that
stereoelectronic effects inherent in the substrate take precedence
in determining the mode of C-O homolytic cleavage even in
the antibody combining site. Furthermore, the observed rate
acceleration could be completely inhibited by preincubation of
the antibody (20 µM) with 1 equiv of the corresponding hapten
(2-acetamidodibenzofuran, 40 µM), highly suggesting that the
catalysis occurs in the antibody combining site. As a further
control, substrate 15 was also irradiated in the presence of BSA,
a protein previously shown to bind small molecules and
accelerate some reactions,21 as well as several nonspecific
antibodies; yet no effect or acceleration on product formation
was observed.
Antibodies for Catalysis of the Photo-Fries Rearrangementa
a (a) glutaric anhydride, diisopropylethylamine, CHCl3, 0 °C. (b) SnCl2,
EtOH, reflux. (c) AcCl, DMAP, pyridine.
kinetics. This is critical as while the occurrence of turnover has
been claimed, it has not been demonstrated on a practical level.
Results and Discussion
Two haptens were designed to resemble points along differing
reaction coordinates. Hapten 12 is a flexible “substrate” and
was made in an attempt to let the combinatorial power of the
immune repertoire sculpt a de noVo pocket such that the formed
radicals could seek either path a or b (Scheme 3). Using this
approach allows for an examination of the interaction of light
with a suitable substrate within the confines of an antibody
combining site where binding energy could alter the energy
surface of the conversion of excited states to the corresponding
photochemical products. Additionally, this type of hapten could
provide an environment such that escape of free radicals from
the solvent cage would be precluded, thereby eliminating the
production of 8 or 11 (Scheme 2). A hapten-substrate approach,
although atypical in relation to other catalytic antibodies, has
proven suitable in the development of antibodies for photo-
chemical reactions as an external source of energy is present in
these reactions.16 In contrast, hapten 13 was designed as a rigid
analogue for the reaction shown in Scheme 2; this hapten is a
planar structure and therefore should elicit complimentary
aromatic residues that could serve as stabilizing moieties upon
the generation of radical species. Additionally, the fortuitous
positioning of a tryptophan residue in the antibody combining
site could also lead to antibody-based sensitization of the photo-
Fries reaction.17b Based upon our recent studies demonstrating
enhanced serum titer with conformationally constrained hap-
tens,19 we felt 13 should also provide an additional model system
for testing this hypothesis in terms of obtaining competent
catalysts.
In accordance with previous observations in our laboratories,19
immunization with conformationally constrained hapten 13 leads
to significantly increased serum titers. Analysis by standard
ELISA techniques of the serum from mice immunized with 13
revealed titers of approximately 128 000-256 000, while im-
munization with hapten 12 gave serum titers of only 8000-
64 000. This clearly gives further credence to the hypothesis
that conformational constraint leads to an elevated immune
response. However, this is not to imply that the use of
conformational constraint in hapten design will lead to increased
catalytic activity.22 Indeed, the most proficient antibodies from
either panel had approximately equal catalytic activity.
Hapten 12 was synthesized in one step from 4-phenoxyaniline
and glutaric anhydride in good yield (50%) (Scheme 3).
Reduction of the nitro group of 2-nitrobenzofuran with tin(II)
chloride, followed by reaction with glutaric anhydride, gave
analogous constrained benzofuran hapten 13 in 11% yield over
two steps. Both haptens were conjugated to keyhole limpet
(20) Kohler, G.; Milstein, C. Nature 1975, 256, 495-497.
(21) Hollfelder, F.; Kirby, A. J.; Tawfik, D. S. Nature 1996, 383, 60-62.
(22) Spivak, D. A.; Hoffman, T. Z.; Moore, A. H.; Taylor, M. J.; Janda, K. D.
Bioorg. Med. Chem. 1999, 7, 1145-1150.
(19) Meijler, M. M.; Matsushita, M.; Altobell, L. J., III; Wirsching, P.; Janda,
K. D. J. Am. Chem. Soc. 2003, 125, 7164-7165.
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A R T I C L E S
Dickerson et al.
Scheme 4. Photo-Fries Rearrangement of 4-Phenoxyaniline
Figure 1. Observed formation of 16 in the presence of antibodies raised
against constrained and unconstrained haptens. The data shown have been
corrected for the background reaction. (b) MT2-21C4, (O) MT4-3G2.
Figure 2. First-order photodecay of photo-Fries biphenyl product 16. ([)
500 µM 16, (9) 400 µM 16, (2) 200 µM 16, (b) 100 µM 16, (]) 40 µM
At low antibody concentrations, the background reaction
dominates the kinetic profile. Thus, kinetic measurements under
standard Michaelis-Menten conditions (i.e., low catalyst con-
centrations relative to substrate) were not possible. Furthermore,
tight binding of the substrate prevented an accurate determina-
tion of Km. In all studied antibodies, the observe rate showed
minimal dependence on the concentration of substrate down to
the lowest concentrations at which product formation could be
detected (10 µM), indicating a Km , 10 µM. Consequently,
examinations of the reaction were performed under set condi-
tions (phosphate-buffered saline, pH 7.4, 2% acetonitrile, 100
µM 15, 20 µM mAb), and initial rates measured. Using an
antibody raised against hapten 13, MT4-3G2, the initial rates
were 8.6 µM/min, while an antibody raised against hapten 12,
MT2-21C4, had an initial product rate of 5.2 µM/min. It was
also noted, under these conditions, the product 16 also presented
itself to be an inactivating photodecay component of the
antibody-mediated process. Interestingly, in the presence of
MT2-21C4, this photoinactivation decay product was clearly
reduced relative to the reaction performed with MT4-3G2
(Figure 1). Presumably, this phenomenon is due to the modi-
fication of a critical combining site residue of MT4-3G2 by
the photodecay product, while the combining site of MT2-21C4
is more robust and able to withstand exposure to this product.
16.
oxygen is well-characterized and of significant interest in the
field of waste water purification.23
Although it has been demonstrated that the antibody structure
is surprisingly resilient to UV irradiation,15 based upon our
kinetic results and the observed reduction in catalytic activity
(vide supra), the ability of the antibodies to bind the substrate
after irradiation was examined. As in previous studies, in the
absence of substrate, no significant loss of binding of either
MT4-3G2 or MT2-21C4 to its antigen was observed after 2 h
of UV irradiation. However, in the presence of substrate, a
pronounced loss in the binding capacity of MT4-3G2 is
observed, as measured by ELISA (Figure 3). Surprisingly, after
only 10 min, a 15% loss in relative binding affinity is observed,
with a 50% loss being observed after 1 h of irradiation. This
supports the notion that the substrate is, in fact, acting in the
combining site of the antibody and that the reaction of interest
also occurs at this site.
The observed decrease in reaction rate can be attributed to
one of two factors, either the produced radicals are covalently
modifying the antibody, thereby serving as mechanism-based
inhibitors, or the photodecay of the recombination product yields
a reactive intermediate which prevents further reaction in the
antibody combining site. Covalent modification of an antibody
combining site is not unprecedented and has been previously
observed in catalytic antibodies for cationic reactions.13 To
assess this difference, MT4-3G2 was irradiated in the presence
of synthetically prepared 16 and then assayed for the ability of
the antibody to bind its hapten. Hapten binding was inhibited
Based upon our kinetic data with MT4-3G2, the photodecay
of 16 was further explored. In aqueous solution, 16 readily
decayed under the high-intensity UV light used for the experi-
ments with an apparent first-order rate constant of 0.11 min-1
(Figure 2). This finding is not particularly surprising as 16
greatly resembles a 1,4-hydroquinone, a known antioxidant.
Furthermore, the reaction of phenolic compounds with singlet
(23) Bonchio, M.; Carofiglio, T.; Carraro, M.; Fornasier, R.; Tonellato, U. Org.
Lett. 2002, 4, 4635-4637.
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Catalysis of Antibody-Mediated Photo-Fries Reaction
A R T I C L E S
Conclusion
Traditionally, the design of haptens for the production of
catalytic antibodies has been grounded in transition state theory
and the stabilization of this transient species. However, it has
recently been appreciated that antibodies are also capable of
altering the energy surface of excited states, thereby stabilizing
highly reactive species and productively channeling their energy
into the appropriate reaction pathways. This study has demon-
strated the ability of an antibody to catalyze the photo-Fries
rearrangement of a diphenyl ether substrate. In the antibodies
studied here, catalysis could be due to multiple factors including
conformational control of the biradical intermediates as well
as the positioning of residues that serve as potential photosen-
sitizers. Despite the presence of significant photodecay of the
product of this reaction and its subsequent inactivation of the
antibody, using a polystyrene resin to trap the photodecay
product, turnover could be observed without using specialized
conditions such as dialysis or “pulsing” the reaction. Further-
more, this photodecay is the limiting factor in the reaction as
the antibody structure was extremely resilient to short-term
irradiation with high-intensity ultraviolet light. Reiterating the
concept of conformational constraint in the elicitation of high
antibody titers,19 a constrained hapten yielded extremely high
immunization responses; although this concept is significant in
the design of new haptens for a number of scenarios, it does
not imply that constraint necessarily leads to higher catalytic
efficiency. Clearly, hapten design with regard to transition state
mimicry of the desired reaction and the induction of essential
catalytic residues remain a challenge in the development of
efficient catalytic antibodies. The results reported expand our
current understanding of antibody catalysts and their catalytic
diversity.
Figure 3. Loss of antibody binding capacity as a result of irradiation in
the presence of photo-Fries substrate and product. (b) 3G2 alone, (9) 3G2
exposed to 15 during irradiation, (2) 3G2 exposed to 16 during irradiation.
Figure 4. Observed turnover of mAb MT4-3G2. The reaction was
performed with 20 µM MT4-3G2 and 200 µM 13 in the presence of an
isocyanate scavenging resin. Data shown are corrected for the background
photochemical reaction.
to an almost identical extent as exposure to irradiated substrate,
implying that photodecay of the reaction product serves as an
inhibitor of the reaction. Analysis of MT4-3G2 after irradiation
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) revealed no visible fragmentation of the antibody
due to the presence of potentially proteolytic radicals or other
reactive species (data not shown).
Experimental Section
General Methods. Unless otherwise stated, all reactions were
performed under an inert atmosphere with dry reagents and solvents
and flame-dried glassware. Analytical thin-layer chromatography (TLC)
was performed using 0.25 mm precoated silica gel Kieselgel 60 F254
plates. Visualization of the chromatogram was by UV absorbance,
iodine, dinitrophenylhydrazine, ceric ammonium molybdate, ninhydrin,
or potassium permanganate as appropriate. Preparative and semi-
preparative TLC was performed using Merck 1 mm or 0.5 mm coated
silica gel Kieselgel 60 F254 plates, respectively. Analytical HPLC was
performed on a Hitachi L-5000 series HPLC using a Vydac 201TP54
reversed phase C18 column. Methylene chloride and chloroform were
distilled from calcium hydride. Tetrahydrofuran (THF) was distilled
from sodium/benzophenone. Methanol was distilled from magenesium.
High-intensity photolysis was performed in a Rayonet photochemical
reactor with RPR-3400 bulbs. Analytical HPLC was performed on a
Hitachi L5000 series HPLC using a Vydac 201TP54 reversed phase
Previous studies using antibodies for photochemical reactions
have focused on observing turnover through a variety of methods
including dialysis of the antibody after the reaction of 1 equiv
of substrate16b and continuously pulsing the reaction with 1 equiv
of substrate and driving the reaction to completion.16a In our
initial studies, turnover could not be directly assessed as
detection was limited by the photodecay of reaction product 16
and the subsequent antibody inactivation (vide supra). To
suppress the inactivation of the antibody, an isocyanate poly-
styrene resin was examined to trap 16 out of the reaction
mixture. Although the resin did not seem to efficiently trap 15,
fortuitously, the resin reacted with the photodecay product,
thereby removing the inhibitory effects of this product. This
effect is surprising as the resin used has a polystyrene backbone
and thus is not expected to appreciably swell in a polar solvent
such as water. Under these conditions, a minimum of 2.5
turnovers could be clearly observed (Figure 4). Presumably, the
entirety of the photodecay product is not removed from the
reaction, thus eventually leading to antibody inactivation and
the observed decrease in rate. Importantly, this represents the
first observation of true turnover in an antibody-catalyzed
reaction involving biradical intermediates.
1
column. H and 13C NMR spectra were recorded on a Bruker AMX-
500 spectrometer at 500 and 125 MHz, respectively, and are reported
in ppm, unless otherwise noted. Matrix-assisted laser desorption/
ionization (MALDI) FTMS experiments are performed on an IonSpec
FTMS mass spectrometer. Electrospray ionization (ESI) mass spec-
trometry experiments were performed on an API 100 Perkin-Elmer
SCIEX single quadrupole mass spectrometer.
4-(4-Phenoxyphenylcarbamoyl)butyric Acid (12). In a dry round-
bottom flask under argon, a cooled (0 °C) solution of 4-phenxoyaniline
(510 mg, 2.8 mmol) in dry CHCl3 (5 mL) was treated successively
with diisopropylethylamine (1.9 mL, 11 mmol) and glutaric anhydride
(628 mg, 5.5 mmol). After 16 h of stirring at room temperature, the
9
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A R T I C L E S
Dickerson et al.
1
reaction mixture was then diluted with one volume of CHCl3. Upon
washing with 1 N HCl, a white precipitate was formed and filtered off
the organic layer. The fluffy solid was dried under high vacuum to
an orange powder (475 mg, 19%). H NMR (CDCl3) 6.98 (d, J ) X
Hz, 1H), 7.40 (m, 3H), 7.57 (m, 2H), 8.09 (m, 1H), 8.18 (s, 1H); 13C
NMR (CDCl3) 117.2, 123.8, 124.6, 125.9, 127.8, 128.2, 129.9, 135.4,
140.2, 153.9. MALDI-FTMS for C12H10NO3 (M + H+) calculated
216.0655, found 216.0654.
1
give 407 mg (50% after crystallization) of the desired hapten 12. H
NMR (DMSO-d6) 1.80 (q, J ) 7.3 Hz, 2H), 2.26 (t, J ) 7.3 Hz, 2H),
2.33 (t, J ) 7.3 Hz, 2H), 6.93 (d, J ) 8.8 Hz, 2H), 6.96 (d, J ) 9.2
Hz, 2H), 7.07 (m, 1H), 7.34 (dd, J ) 8.8 Hz, 7.3 Hz, 2H), 7.59 (d, J
) 9.2 Hz, 2H), 9.91 (s, 1H); 13C NMR (DMSO-d6) 20.5, 33.0, 35.4,
117.8, 119.5, 120.7, 122.9, 130.0, 135.3, 151.4, 157.5, 170.6, 174.2.
MALDI-FTMS for C17H18NO4 (M + H+) calculated 300.1230, found
300.1229.
2-Hydroxy-5-aminobiphenyl (16). 2-Hydroxy-5-nitrobiphenyl (25
mg, 0.12 mmol) was dissolved in methanol (30 mL), and Pd/C (5%, 5
mg) was added. The reaction was then shaken on a Parr shaker with
hydrogen (40 psi) for 2 h. The crude mixture was then passed through
a pad of Celite and concentrated to give the desired compound (21
mg, 98%). 1H NMR (CDCl3) 6.78 (m, 3H), 7.29 (m, 3H), 7.54 (s, 2H);
13C NMR (CDCl3) 116.6, 117.9, 126.2, 128.4, 131.9, 142.6, 153.8.
MALDI-FTMS for C12H12NO (M + H+) calculated 186.0913, found
186.0914.
2-Hydroxy-5-acetamidobiphenyl. 2-Hydroxy-5-aminobiphenyl (150
mg, 0.81 mmol) was dissolved in CH2Cl2 (10 mL), and DIEA (157
mg, 212 µL, 1.22 mmol) was added. The mixture was cooled to 0 °C,
acetyl chloride (70 mg, 63 µL, 0.89 mmol) was slowly added, and the
reaction was stirred 12 h at room temperature. The crude diacetyl
product was then treated with a solution of 1 M NaOH and methanol
(1:3; 10 mL) and stirred for an additional 2 h. The reaction was then
concentrated and purified by preparative TLC (5% MeOH/CH2Cl2) to
yield 114 mg of the desired product (62%). 1H NMR (CDCl3) 2.15 (s,
3H), 6.82 (m, 1H), 7.35 (m, 5H), 7.57 (m, 2H); 13C NMR (CDCl3)
24.1, 114.2, 118.9, 122.2, 125.4, 128.1, 129.5, 134.2, 136.7, 152.1,
168.2. MALDI-FTMS for C14H14NO2 (M + H+) calculated 228.1019,
found 228.1015.
4-(Dibenzofuran-2-ylcarbamoyl)butyric Acid (13). 2-Aminod-
ibenzofuran (23 mg, 0.13 mmol) was dissolved in CHCl3 (2 mL) and
cooled to 0 °C. This solution was then treated successively with
diisopropylethylamine (88 µL, 0.5 mmol) and glutaric anhydride (29
mg, 0.25 mmol). After 3 h at room temperature, the reaction mixture
was then diluted with one volume of CHCl3. Upon washing with 1 N
HCl, a white precipitate was formed and filtered off the organic layer.
The organic layer was then concentrated under reduced pressure and
the residue combined to the solid obtained by filtration. The crude
material was purified by successive preparative TLC eluting first with
EtOAc/hexane/AcOH (4:4:0.8) followed by toluene/EtOAc/AcOH (7:
3:0.5) to give 9 mg (24%) of the desired compound as an off-white
1
solid. H NMR (CD3OD) 2.02 (q, J ) 7.3 Hz, 2H), 2.35 (t, J ) 7.3
Hz, 2H), 2.46 (t, J ) 7.3 Hz, 2H), 7.93 (td, J ) 7.8 Hz, 1.5 Hz, 1H),
7.46 (td, J ) 7.8 Hz, 1.5 Hz, 1H), 7.52 (m, 2H), 7.56 (d, J ) 8.0 Hz,
1H), 7.98 (d, J ) 8.5 Hz, 1H), 8.31 (s, 1H); 13C NMR (CD3OD) 21.3,
33.4, 37.2, 104.8, 111.9, 113.6, 118.2, 121.6, 124.1, 125.7, 128.3, 135.5,
147.1, 155.6, 172.0, 177.4. MALDI-FTMS for C17H16NO4 (M + H+)
calculated 298.1074, found 298.1066.
4-Amino-4′-hydroxybiphenyl (19). 4-Nitro-4′-hydroxybiphenyl (100
mg, 0.47 mmol) was dissolved in ethyl acetate (40 mL), and palladium
on carbon was added (10%, 10 mg). The resulting suspension was then
hydrogenated at 36 psi for 18 h at room temperature. The reaction was
then filtered through a cake of Celite and concentrated to yield a yellow-
4-Phenoxyacetanilide (14). To a cooled solution (0 °C) of 4-phe-
noxyaniline (570 mg, 4.05 mmol) in pyridine (4 mL) under argon, acetyl
chloride (864 µL, 12 mmol), and N,N-dimethylaminopyridine (50 mg,
0.4 mmol) were added successively. The reaction was allowed to stir
at room temperature for 16 h, after which time EtOAc was added and
the organic layer washed several times with aqueous 1 N HCl and brine
and then dried over MgSO4. Purification of the crude material by flash
chromatography on SiO2 eluting with hexane/EtOAc (3:2) gave 625
1
brown solid (81 mg, 93%). H NMR (CDCl3) 6.68 (m, 4H), 7.28 (m,
4H); 13C NMR (CDCl3) 115.4, 116.2, 126.0, 127.9, 129.6, 139.3, 145.8,
158.5. MALDI-FTMS for C12H12NO (M + H+) calculated 186.0913,
found 186.0911.
4-N-Acetamido-4′-hydroxybiphenyl. 4-Amino-4′-hydroxybiphenyl
(89 mg, 0.48 mmol) was dissolved in THF (50 mL), and DIEA (93
mg, 126 µL, 0.72 mmol) was added. The mixture was cooled to 0 °C,
acetyl chloride (42 mg, 38 µL, 0.53 mmol) was slowly added, and the
reaction was stirred 12 h at room temperature. The crude diacetyl
product was then treated with a solution of 1 M NaOH and methanol
(1:3; 10 mL) and stirred for an additional 2 h. The reaction was then
concentrated and purified by preparative TLC (5% MeOH/CH2Cl2) to
1
mg (68%) of the desired material as a white solid. H NMR (CDCl3)
2.14 (s, 3H), 6.95 (m, 4H), 7.08 (t, J ) 7.3 Hz, 1H), 7.31 (dd, J ) 8.5
Hz, 7.6 Hz, 2H), 7.45 (d, J ) 8.8 Hz, 1H), 8.00 (br s, 1H); 13C NMR
(CDCl3) 24.8, 118.3, 119.4, 121.8, 123.0, 129.7, 133.4, 153.4, 157.4,
168.8. MALDI-FTMS for C14H14NO2 (M + H+) calculated 228.1019,
found 228.1017.
1
yield 63 mg of the desired product (58%). H NMR (CDCl3) 2.38 (s,
4-N-Acetamido-2′-hydroxybiphenyl. To a solution of 2-iodophenol
(220 mg, mmol) in water (10 mL), 4′-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)acetanilide (261 mg, mmol), K2CO3 (415 mg, mmol),
and 10% palladium on carbon (10 mg) were added. The reaction mixture
was stirred for 12 h vigorously and then quenched slowly with 1.5 M
HCl (10 mL). The solution was then filtered to isolate a tan solid along
with the catalyst. The solid was then triturated with ethyl acetate, and
the resulting solution was concentrated to yield 200 mg (88%) of the
3H), 7.19 (m, 4H), 7.62 (m, 4H); 13C NMR (CDCl3) 24.5, 116.8, 120.6,
124.3, 125.8, 132.6, 142.0, 145.3, 158.6, 169.2. MALDI-FTMS for
C14H14NO2 (M + H+) calculated 228.1019, found 228.1021.
Preparation of Immunoconjugates. Hapten (5 mg) was treated with
1.3 equiv of 3-sulfo-N-hydroxysuccinimide and 1.3 equiv of 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride for 24 h in
DMF. This reaction was then added to solutions (5 mg/mL) of keyhole
limpet hemocyanin (KLH) and bovine serum albumin (BSA) in PBS
(50 mM, pH 7.4), respectively, and allowed to react 24 h at 4 °C.
Hybridoma Production. See ref 21.
Kinetic Assays. Antibody stock concentrations were determined by
UV-vis spectroscopy using 1 OD ) 1.25 mg/mL at 280 nm and 1
mg/L ) 6.7 µM. The assay was initiated by the addition of a solution
of antibody (20 µM in PBS) to a solution of 4-phenoxyaniline 13 (200
µM) in buffer (10 mM PBS, pH 7.4) with 2% acetonitrile in 4 mL
glass vials. High-intensity photolysis was performed for 30 min
intervals, and at times throughout the assay, aliquots (20 µL) were
removed and directly injected onto a RP-C18 Vydac HPLC column,
equipped with a guard column (HPLC conditions, Isocratic mobile phase
of acetonitrile/water (20:80) with 0.1% TFA. Solvent flow rate of 1
mL min-1 and detection at 254 nm). Biphenyl product 16 (retention
1
desired compound. H NMR (CDCl3) 2.22 (s, 3H), 6.97 (d, J ) 8.1
Hz, 2H), 7.22 (m, 2H), 7.44 (d, J ) 8.4 Hz, 2H), 7.61 (d, J ) 8.4 Hz,
2H); 13C NMR (CDCl3): 24.3, 116.7, 122.5, 129.6, 134.4, 142.7, 151.4,
169.3. MALDI-FTMS for C14H14NO2 (M + H+) calculated 228.1019,
found 228.1022.
2-Hydroxy-5-nitrobiphenyl. 2-Hydroxybiphenyl (2 g, 11.8 mmol)
was dissolved in glacial acetic acid (50 mL) and cooled to 0 °C. To
this solution, concentrated nitric acid (680 µL) in glacial acetic acid
(10 mL) was added dropwise via addition funnel. The reaction was
then warmed to room temperature and stirred vigorously for 2 h.
Saturated aqueous NaHCO3 (300 mL) was then added slowly, and the
aqueous layer was extracted with CH2Cl2 (3 × 200 mL). The combined
organic layers were then dried on MgSO4 and concentrated. Purification
on silica with EtOAc/hexane (1:9) yielded the desired compound as
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15400 J. AM. CHEM. SOC. VOL. 125, NO. 50, 2003

Catalysis of Antibody-Mediated Photo-Fries Reaction
A R T I C L E S
time ) 5.2 min) was determined by interpolation of peak height and
area values relative to standard curves.
for 30 min. The plate was developed with the colorimetric reagent
ABTS for 30 min, and the absorbance was measured on an ELISA
plate reader at 405 nm.
ELISA Binding Experiments. An ELISA plate (CoStar; 96-well)
was coated uncovered overnight at 37 °C with 25 µL of the MT4-BSA
conjugate diluted to 5 µg/mL. To this, methanol (50 µL/well) was added
and allowed to sit 5 min at room temperature. The methanol was then
removed from the wells, and the plates were allowed to dry. Wells
were then blocked with 50 µL of Blotto (5% skim milk powder in
PBS) for 10 min at 37 °C. Typically, 25 µL of a 1:1000 dilution of
mAbMT4-3G2 in Blotto were then added and incubated 1 h at 37 °C.
After washing, 25 µL of a 1:1000 dilution of a goat-anti-mouse/
horseradish peroxidase conjugate in Blotto were added and incubated
Acknowledgment. This work was supported financially by
The Scripps Research Institute, The Skaggs Institute for
Chemical Biology, and the National Institutes of Health (GM-
43858 to K.D.J.). We thank Dr. Anita Wentworth for helpful
discussions related to this work.
JA038016N
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J. AM. CHEM. SOC. VOL. 125, NO. 50, 2003 15401